This subunit is homolog of S. cerevisiae SGF73 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf73]] subunit of SAGA and deletion of this subunit in S. cerevisiae results in viable cells but no induction of PAU genes under hypoxic conditions.

This subunit is homolog of S. cerevisiae SGF73 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf73]] subunit of SAGA and deletion of this subunit in S. cerevisiae results in viable cells but no induction of PAU genes under hypoxic conditions.

DNA Reagents

had DNA 2.0 codon-optimize S. pombe gene SPCC126.04c [[4]]
including biobrick ends for expression of ORF in S. cerevisiae
This subunit is homolog of S. cerevisiae SGF73 [[5]] subunit of SAGA and deletion of this subunit in S. cerevisiae results in viable cells but no induction of PAU genes under hypoxic conditions.

What I'd like to try is to codon optimize this protein for expression
in S. cerevisiae (nuclear expression, not mitochondrial as before!).
Again this ORF can have none of the "forbidden" sites (EcoRI, XbaI, NotI, SpeI or PstI ) that are used for cloning. Once it's been
optimized the cloning sites can be added by tagging the 5' end before
the ATG with: GTTTCTTCGAATTCGCGGCCGCTTCTAGAG and tagging the 3' end after the stop with: TACTAGTAGCGGCCGCTGCAGGAAGAAAC.

Primers

sgf73::KanMX_KO_fwd (JW109)

5' TGAACACACAAGAGAAGCGCAAAAGAGTAAAGAGCTAAA ctgtgcggtatttcacaccg

sgf73::KanMX_KO_rev (JW110)

5' CTCACTTCGTGAACATGCTGGATAACGTGCATGATTCAA agattgtactgagagtgcac

sgf73_URA3_KO_fwd (NO99)

5' TGAACACACAAGAGAAGCGCAAAAGAGTAAAGAGCTAAAatgtcgaaagctacatataa

sgf73_URA3_KO_rev (NO100)

5' CTCACTTCGTGAACATGCTGGATAACGTGCATGATTCAA ttagttttgctggccgcatc

sgf29_URA3_KO_fwd (NO101)

5' GACTTTTTCACAGCAAAACACACGGTCACCTTTCTTATTatgtcgaaagctacatataa

sgf29_URA3_KO_rev (NO102)

5' AGAAGATCTTATGATATGTAGTAAATGTTAACCACCATTttagttttgctggccgcatc

SGF73_100upstm_fwd (NO107)

5'- CCA GCA TCG CTC ATT AGA GAT TAA TTT

SGF73_100dwstm_rev (NO108)

5'- GCG TAA CTT GTC CAC AAA CAT AGA CGT G

SGF29_100upstm_fwd (NO109)

5'- CGA TTT TGC CAG ATA CGA AAA AGG TTA AAG AC

SGF29_100dwstm_rev (NO110)

5'- CAC CTT CAA GTG AAT ACA TCA CTT CCG ACG

URA3_464bpDwstATG_fwd (FO3672 =MH243) Tm 58.8

5'- GCAAGGGCTCCCTAGCTACT

URA3_638bpDwstATG_rev (FO3673 =MH244) Tm 55

5'- AATGCGTCTCCCTTGTCATC

SpSGF73*toSc_fwd (where * indicates codon optimized version of the Sp homolog of the Sc SGF73) (NO111)

MH176=hypoxic gene expression reporter strain

related email note 07.12.06 from Mark
Natalie,
I only have PAU5-URA3 or DAN3-HIS3. The DAN3 reporter is tighter on plates, perhaps
because the His phenotype is tighter in general. I have not checked whether SGF73 is
required for PAU5-URA3 or DAN3-HIS3 induction specifically. But I do know that in an
sgf73 delta mutant, there is no hypoxic PAU expression. So I infer that PAU5 and DAN3
expression is affected.
We can talk in the afternoon.
Mark

Phenotype MH176 (~wt) v FY2475 (sgf73::KanMX) v FY2474 (sgf29::KanMX)

First spot where individual colonies seen is listed
Better wt control would be FY3 since FY strains are all hap1

Plate type

Temp

MH176 (~wt)

FY2475 (sgf73::KanMX)

FY2474 (sgf29::KanMX)

comments

YPD

30° 3d

6

6

6

Mark has seen 73 KO grow more slowly doing growth curve

YPD

37° 3d

6

6

6

YPD

14° 4d

4

4

4

YPgal

30° 3d

6

4 (repeat this?)

6

YPgal

37° 3d

5

5

5

YPgal

14° 4d

2

2

2

YPKAc

30° 3d

5

5

5

YPKAc

37° 3d

5

5

5

YPKAc

14° 4d

4

4

4

YPEG

30° 3d

3-4

2-3

2-3

YPEG

37° 3d

2-3

1-2

1-2

YPEG

30° 4d

6

3

3-4

YPEG

37° 4d

3

2

2

greater diff. seen at 30° on YPEG

Phenotype wt vs sgf73 vs sgf29 vs double

First spot where individual colonies seen is listed
FY3, FY2475, FY2474 and NY356 all FY bkgd so hap1
MH176, NY350 in HAP1 bkgd and carry dan3::HIS3 reporter

Plate type

Temp

FY3 (~wt)

MH176 (~wt)

FY2475 (sgf73::KanMX)

NY350 (sgf73::URA3 in MH176)

FY2474 (sgf29::KanMX)

FY356 (sgf29::URA3 sgf73::KanMX)

comments

YPD

30° 4d

5-6

5-6

5-6

5-6

5-6

5-6

double mutant grows more slowly but same spot # comes up

YPD +1M NaCl

30° 4d

5

5

5-6

5-6

5-6

5-6

no NaCl p-type at 37° or 14° either

YPD +3% formamide

30° 4d

5-6

5-6

3-4

3-4

5-6

1

sgf73 alone has some sensitivity to formamide and sgf29, sgf73 has much greater sensitivity (sgf29 alone has ~no sensitivity). Can use this synthetic phenotype as well as 73 phenotype to look for complementation by pombe subunit (underway 08.02.06). Note: 37° and formamide suppresses the sgf73 phenotype but synthetic phenotype of double still evident. 14° and formamide had showed sensitivity for sgf73::KanMX but not sgf73::URA3...need to recheck this since v. strange.

YPEG

30° 4d

4-5

4-5

2-3

2-3

4-5

v little growth even on 1

effect mimics what is seen on YPD +3%formamide. Can use this for complementation tests as well. Not known why NFCS ~lethal to sgf73, sgf29 double mutant.

YPgal

30° 3d

5-6

5-6

4-5

4-5

5-6

3-4

result recapitulates what was seen before but effects not as dramatic as on YPD +3% formamide or YPEG